Abstract Carbon monoxide (CO) is an endogenous signaling molecule with importance on par with nitric oxide (NO), the subject of the 1998 Nobel Prize. It is produced from heme degradation by heme oxygenases. Extensive literature reports have convincingly demonstrated the therapeutic effects of CO as an anti-inflammatory agent in models of colitis, sepsis, liver injury, and organ transplant. While much is known regarding the efficacy of CO, the challenge now is to develop pharmaceutically acceptable deliverable forms of CO as research tools and possible therapeutics. Inhaled CO has been the major form of delivery in most preclinical work. However, this is not the ideal modality because of difficulties in safe administration and in controlling doses, lack of portability, and the dependence on each individual patient?s respiratory function to deliver precise amounts. There have also been a number of metal-based CO-releasing molecules (CO-RMs) and photo-sensitive organic CO-RMs. However, metal toxicity and light accessibility issues impose limitations. In an exciting development, we have developed several classes of organic CO-prodrugs with tunable release rates. We propose to explore the potential of using such prodrugs to treat inflammation and tissue injury using experimental colitis (EC) in mice as a model. Others and we have demonstrated the unique ability of CO to reduce inflammation, promote tissue repair, and enhance host defense against pathogenic bacterial infection. Therefore, CO has enormous potential to be an effective treatment for colitis without the increased risk of infection associated with broad immunosuppression. With the high cost of drug discovery and development which is way beyond the funds available in an NIH application, we plan to initially examine a well-defined set of issues. The availability of prodrugs with tunable release rates offers the opportunity for the first time to examine the interplay among dosage, efficacy, pharmacokinetics, and release profiles, which is a unique problem with a gasotranmistter. In this MPI application, we combine the extensive expertise of the Wang, Tan, and Otterbein labs, and propose to build on compelling preliminary data to develop organic CO-prodrugs for treating inflammation in EC models. Our central hypothesis is that CO-prodrugs acts therapeutically in EC by modulating the intestinal microenvironment. We will test this with the following 2 specific aims: 1.) synthesize, optimize, and assess CO prodrugs; and 2.) evaluate the CO-prodrugs in EC. Our preliminary results clearly show efficacy of such CO-prodrugs in treating murine EC, sepsis, and liver injury. Upon completion of the project, we expect to have: 1.) developed a series of CO prodrugs, 2.) demonstrated the feasibility of using such prodrugs to treat inflammation in EC models; and (3) defined the relationship among dose, release kinetics, pharmacokinetics and efficacy with a clear therapeutic window. The clinical potential of CO-based therapeutics as anti-inflammatory agents is profound and could impact other areas such as organ transplantation, stroke, and heart attack.